Advertisement

Acta Parasitologica

, Volume 59, Issue 1, pp 132–138 | Cite as

A PCR method targeting internal transcribed spacers: the simultaneous detection of Babesia bigemina and Babesia bovis in cattle

  • Junlong Liu
  • Guiquan Guan
  • Aihong Liu
  • Youquan Li
  • Hong Yin
  • Jianxun Luo
Original Paper

Abstract

In this study, two pairs of oligonucleotide primers were designed according to the nucleotide sequence of the internal transcribed spacers (ITSs) of Babesia bigemina and B. bovis isolates from China. The primers were used in a multiplex PCR to detect parasite DNA in blood samples from cattle. There was no cross reactions with B. ovata, B. major, B. sp. Kashi, Theileria annulata, T. sergenti, T. sinensis or normal bovine DNA. The sensitivity of multiplex PCR assay was 1 pg and 10 pg DNA for B. bigemina and B. bovis, respectively. A total of 260 field blood samples collected from cattle in five provinces of China were analyzed by multiplex PCR and light microscopy. PCR testing revealed that 7.3% (19/260) and 5.8% (15/260) of cattle were positive for B. bigemina and B. bovis and 1.2% (3/260) of cattle were co-infected with B. bigemina and B. bovis. Using light microscopy, 2.3% (6/260) and 1.5% (4/260) of cattle were infected by B. bigemina and B. bovis, respectively, and no co-infection was found. The results showed that the multiplex PCR developed in the present study could be an alternative diagnostic tool for the detection of B. bovis and B. bigemina infection in cattle.

Keywords

Babesia bigemina Babesia bovis Multiplex PCR ITS ribosomal DNA 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aktas M., Bendele K.G., Altay K., Dumanli N., Tsuji M., Holman P.J. 2007. Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species. Veterinary Parasitology, 147, 221–230. DOI: 10.1016/j.vetpar.2007.04.007.PubMedCrossRefGoogle Scholar
  2. Alhassan A., Pumidonming W., Okamura M., Hirata H., Battsetseq B., Fujisaki K., Yokoyama N., Igarashi I. 2005. Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood. Veterinary Parasitology, 129, 43–49. DOI: 10.1016/j.vetpar.2004.12.018.PubMedCrossRefGoogle Scholar
  3. Allred D.R. 2003. Babesiosis: persistence in the face of adversity. Trends in Parasitology, 19, 51–55.PubMedCrossRefGoogle Scholar
  4. Almeria S., Castellà J., Ferrer D., Ortuño A., Estrada-Peña A., Gutiérrez J.F. 2001. Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection. Veterinary Parasitology, 99, 249–259.PubMedCrossRefGoogle Scholar
  5. Baneth G., Kenny M.J., Tasker S., Anug Y., Shkap V., Levy A., Shaw S.E. 2004. Infection with a proposed new subspecies of Babesia canis, Babesia canis subsp. present in domestic cat. Journal of Clinical Microbiology, 42, 99–105.PubMedCentralPubMedCrossRefGoogle Scholar
  6. Barros S.L., Madruga C.R., Araujo F.R., Menk C.F., Almeida M.A., Melo E.P.S., Kessler R.H. 2005. Serological survey of Babesia bovis, Babesia bigemina and Anaplasma marginale antibodies in cattle from the semi-arid region of Bahia, Brazil, by enzyme linked immunosorbent assays. Memórias do Instituto Oswaldo Cruz, 100, 513–517. DOI:/S0074-02762005000600003.PubMedCrossRefGoogle Scholar
  7. Bishop J.P., Adams L.G. 1973. Combination thin and thick blood films for the detection of Babesia parasitemia. American Journal of Veterinary Research, 34, 1213–1214.PubMedGoogle Scholar
  8. Conrad P.A., Kjemtrup A.M., Carreno R.A., Thomford J., Wainwright K., Eberhard M., Quick R., Telford S.R.3rd, Herwaldt B.L. 2006. Description of Babesia duncani n. sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms. International Journal for Parasitology, 36, 779–789. DOI: 10.1016/j.ijpara.2006.03.008.PubMedCrossRefGoogle Scholar
  9. Figueroa J.V., Chieves L.P., Johnson G.S., Buening G.M. 1993. Multiplex polymerase chain reactionbased assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Veterinary Parasitology, 50, 69–81.PubMedCrossRefGoogle Scholar
  10. Fu Y.B., Lai Z.S. 1997. A report of buffalo babesiosis. Guangxi Journal of Animals Husbandry Veterinary Medicine, 3, 46–48 (In Chinese).Google Scholar
  11. García-Sanmartín J., Nagore D., García-Pérez A.L., Juste R.A., Hurtado A. 2006. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays. BMC Veterinary Research, 2, 16. DOI: 10.1186/1746-6148-2-16.PubMedCentralPubMedCrossRefGoogle Scholar
  12. Gubbels J.M., de Vos A.P., van der Weide M., Viseras J., Schouls L.M., de Vries E., Jongejan F. 1999. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. Journal of Clinical Microbiology, 37, 1782–1789.PubMedCentralPubMedGoogle Scholar
  13. Holman P.J., Bendele K.G., Schoelkopf L., Jones-Witthuhn R.L., Jones S.O. 2003. Ribosomal RNA analysis of Babesia odocoilei isolates from farmed reindeer (Rangifer tarandus tarandus) and elk (Cervus elaphus canadensis) in Wisconsin. Parasitology Research, 91, 378–383. DOI: 10.1007/s00436-003-0984-5.PubMedCrossRefGoogle Scholar
  14. Kho W.G., Chung J.Y., Sim E.J., Kim M.Y., Kim D.W., Jongwutiwes S., Tanabe K. 2003. A multiplex polymerase chain reaction for the differential diagnosis of Plasmodium falciparum and Plasmodium vivax. Parasitology International, 52, 229–236.PubMedCrossRefGoogle Scholar
  15. Kim C.M., Blanco L.B.C., Alhassan A., Iseki H., Yokoyama N., Xuan X., Igarashi I. 2008. Development of a Rapid Immunochromatographic Test for Simultaneous Serodiagnosis of Bovine Babesioses caused by Babesia bovis and Babesia bigemina. American Journal of Tropical Medicine and Hygiene, 78, 117–121.PubMedGoogle Scholar
  16. Liu A., Guan G., Du P., Gou H., Liu Z., Liu J., Ma M., Yang J., Li Y., Niu Q., Ren Q., Bai Q., Yin H., Luo J. 2012. Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina. Parasitology International, 61, 658–663. DOI: 10.1016/j.parint.2012.07.004.PubMedCrossRefGoogle Scholar
  17. Liu J.L., Yin H., Liu G.Y., Guan G.Q., Ma M.L., Liu A.H., Liu Z.J., Li Y.Q., Ren Q.Y., Dang Z.S., Gao J.L., Zhao H.P., Luo J.X. 2008. Discrimination of Babesia major and Babesia ovata based on ITS1-5.8S-ITS2 region sequences of rRNA gene. Parasitology Research, 102, 709–713. DOI: 10.1007/s00436-007-0818-y.PubMedCrossRefGoogle Scholar
  18. Markoulatos P., Siafakas N., Moncany M. 2002. Multiplex polymerase chain reaction: a practical approach. Journal of Clinical Laboratory Analysis, 16, 47–51.PubMedCrossRefGoogle Scholar
  19. McCosker P.J. 1981. The global importance of babesiosis. In: Ristic M., Kreier J.P. (Eds.), Babesiosis. Academic Press, New York, NY, 24 pp.Google Scholar
  20. Mclaughlin G.L., Edlind T.D., Ihler G.M. 1986. Detection of Babesia bovis using DNA hybridization. Journal of Protozoology, 33, 125–128.PubMedCrossRefGoogle Scholar
  21. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, E63.PubMedCentralPubMedCrossRefGoogle Scholar
  22. Oliveira M.C.S., Oliveira-Sequeira T.C.G., Regitano L.C.A., Alencar M.M., Néo T.A., Silva A.M., Oliveira H.N. 2008. Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick. Veterinary Parasitology, 155, 281–286. DOI: 10.1016/j.vetpar.2008.04.022.PubMedCrossRefGoogle Scholar
  23. Passos L.M., Bell-Sakyi L., Brown C.G. 1998. Immunochemical characterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina. Veterinary Parasitology, 76, 239–249.PubMedCrossRefGoogle Scholar
  24. Qin W.X. 2005. Diagnosis and therapy of bovine babesiosis. Today Animal Husbandry and Veterinary Medicine, 8, 10 (in Chinese).Google Scholar
  25. Salih D.A., El Hussein A.M., Seitzer U., Ahmed J.S. 2007. Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan. Parasitology Research, 101, 1035–1044. DOI: 10.1007/s00436-007-0583-y.PubMedCrossRefGoogle Scholar
  26. Taylor M.A., Coop R.L., Wall R.L. 2007. Veterinary Parasitology. Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK, 104–105.Google Scholar
  27. Tenter A.M., Friedhoff K.T. 1986. Serodiagnosis of experimental and natural Babesia equi and B. caballi infections. Veterinary Parasitology, 20, 49–61.PubMedCrossRefGoogle Scholar
  28. Yang Z.C. 1984. A research concerning a four foci babesiosis outbreak. Veterinary Science in China, 4, 36–37 (in Chinese).Google Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2014

Authors and Affiliations

  • Junlong Liu
    • 1
  • Guiquan Guan
    • 1
  • Aihong Liu
    • 1
  • Youquan Li
    • 1
  • Hong Yin
    • 1
  • Jianxun Luo
    • 1
  1. 1.State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research InstituteChinese Academy of Agricultural ScienceLanzhou, GansuP. R. China

Personalised recommendations